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Synthesis of carbon-11-labeled tariquidar derivatives as new PET agents for imaging of breast cancer resistance protein (ABCG2)
ARTICLE IN PRESS Applied Radiation and Isotopes 68 (2010) 1098–1103
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Synthesis of carbon-11-labeled tariquidar derivatives as new PET agents for imaging of breast cancer resistance protein (ABCG2) Min Wang a, David X. Zheng a, Michael B. Luo a, Mingzhang Gao a, Kathy D. Miller b, Gary D. Hutchins a, Qi-Huang Zheng a, a b
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, L3-208, Indianapolis, IN 46202, USA Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e in f o
a b s t r a c t
Article history: Received 20 November 2009 Received in revised form 29 January 2010 Accepted 6 February 2010
Carbon-11-labeled tariquidar derivatives were first designed and synthesized as new PET agents for imaging of breast cancer resistance protein. The target tracers were prepared by O-[11C]methylation of their corresponding acid precursors using [11C]CH3OTf under basic conditions and isolated by a simplified solidphase extraction (SPE) method in 50–60% radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 15–20 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 111–185 GBq/mmol. & 2010 Elsevier Ltd. All rights reserved.
Keywords: Positron emission tomography Breast cancer resistance protein Tariquidar derivatives Radiotracers Imaging
1. Introduction Breast cancer is the most common cancer diagnosed in women and is the second leading cause of death in women after lung cancer in the United States (Wang et al., 2009). Breast cancer resistance protein 2, a member of the G subfamily of the ATP-binding cassette (ABC) transporter superfamily (ABCG2) which transports a wide variety of substrates, is an attractive target for the development of therapeutic agents for use in breast cancer treatment, since ABCG2 plays an important role in conjunction with multidrug resistance (MDR) and is associated with a new concept of tumor development ¨ and progression (cancer stem cell hypothesis) (Kuhnle et al., 2009). Recently a new series of tariquidar derivatives have been developed as potent and selective inhibitors of ABCG2, and the representative compounds methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino)benzoate (6a), methyl 4-((4-(2-(6,7-dimethoxy-1,2,3, 4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3-carbonylamino)benzoate (6b), methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-6-carbonylamino)benzoate (6c) and methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoxaline-2-carbonylamino)benzoate (6d) exhibited excellent biological activity with nanomolar IC50
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E-mail address: [email protected] (Q.-H. Zheng). 0969-8043/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2010.02.008
values as 60, 119, 179 and 183 nM for ABCG2, respectively, and about 100–500 fold selectivity for ABCG2 over other efflux transport proteins such as a member of the B subfamily of the ABC transporter superfamily (ABCB1, p-glycoprotein 170 or multidrug resistance related protein 1) and a member of the C subfamily of the ABC transporter superfamily (ABCC2, multidrug resistance related ¨ protein 2) (Kuhnle et al., 2009). Most breast cancer patients will survive after conventional treatment modalities such as surgery, radiation therapy, hormone therapy, chemotherapy and/or a combination thereof if the breast cancer can be detected at the early stage (Barry and Kell, 2009). However, these treatments still fail to cure a significant number of patients. Rapid and accurate early detection is highly desirable so that various therapeutic regiments can be given before the primary tumors become widely spread to other organs (Liu et al., 2007). In the diagnosis and treatment of breast cancer, the biomedical imaging technique positron emission tomography (PET) has become a clinically valuable and accepted new medical imaging tool (Pons et al., 2009). PET combining with radiopharmaceutical 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) is widely used in clinical settings to diagnose breast cancer (Escalona et al., 2009). [18F]FDG is the only PET breast cancer imaging agent used clinically at this point in time. However, [18F]FDG is not in all cases satisfactory. [18F]FDG as a PET tracer has limitations including the inability to visualize very small size breast tumors in early stage and the low cellular uptake rate in breast cancer (Czernin, 2002). In addition, only a limited number of PET studies using other radiotracers have been conducted in clinical research to image breast cancer and monitor its response to treatment, due to the
ARTICLE IN PRESS M. Wang et al. / Applied Radiation and Isotopes 68 (2010) 1098–1103
limited accessibility of radiotracers. These limitations have motivated the development of new breast cancer PET imaging agents. Radiotracer development is a key area for advancement of research and clinical applications of PET in cancer diagnosis and treatment (Fowler et al., 1999). ABCG2 is an attractive imaging target for PET early detection of breast cancer in patient. Our hypothesis is that tariquidar derivatives labeled with a positron emitting radionuclide carbon-11 may enable non-invasive monitoring of ABCG2 expression in breast cancer and breast cancer response to ABCG2 inhibitor therapy using PET imaging techniques. To radiolabel ABCG2 therapeutic agents as diagnostic agents, we have first designed and synthesized carbon-11-labeled tariquidar derivatives, 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquino[11C]methyl line-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino)benzoate ([11C]6a), [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3, 4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3-carbonylamino)benzoate ([11C]6b), [11C]methyl 4-((4-(2(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl) aminocarbonyl)-2-(quinoline-6-carbonylamino)benzoate ([11C]6c) and [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoxaline-2-carbonylamino)benzoate ([11C]6d), as new PET agents for imaging of ABCG2. 2. Results and discussion 2.1. Chemistry Four title compounds 6a–d were served as the reference standards and selected for radiolabeling. These tariquidar derivatives are potent
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and selective ABCG2 inhibitors with the O-methyl group. This provides position for making carbon-11 tracers with [11C]methyl iodide ([11C]CH3I) or [11C]methyl triflate ([11C]CH3OTf) (Jewett, 1992; Mock et al., 1999), which could serve as PET agents. Therefore, these compounds can be proposed as tools for PET experiments. The purpose of this study is to convert therapeutic agents to diagnostic agents. The target compounds 6a–d and their corresponding acid precursors, 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino) benzoic acid (7a), 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3-carbonylamino)benzoic acid (7b), 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-6-carbonylamino)benzoic acid (7c) and 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoxaline-2-carbonylamino)benzoic acid (7d), were synthesized according to the procedures outlined in Scheme 1. Synthesis of the reference standards was based on the literature methods described ¨ previously (Hubensack, 2005; Kuhnle et al., 2009) with slight modifications. Isoquinoline 1 was obtained by the Pictet– Spengler reaction of 2-(3,4-dimethoxyphenyl)ethylamine with paraformaldehyde in formic acid in 95% yield (Okano et al., 2006). Subsequent nucleophilic substitution of compound 1 with 4-nitrophenethyl bromide in the presence of K2CO3 in DMF provided 6,7-dimethoxy-2-(4-nitrophenethyl)-1,2,3,4tetrahydroisoquinoline (2) in 64% yield. Reduction of nitro group of compound 2 was performed efficiently with SnCl2 and concentrated HCl instead of hydrogenation under high pressure. Using this method, it afforded 4-(2-(6,7-dimethoxy-1,2,3,
Scheme 1. Synthesis of tariquidar derivative standards (6a–d) and precursors (7a–d). Reagents and conditions: (a) (CH2O)n, HCO2H, 50 1C, overnight; (b) 4-nitrophenethyl bromide, K2CO3, DMF, 100 1C, 8 h; (c) SnCl2, HCl, MeOH, 90 1C, 5 h; (d) 3-nitro-4-carbomethoxybenzyl chloride, triethylamine, CH2Cl2, 50 1C, 4 h; (e) H2, 10% Pd/C, THFMeOH, RT, 7 h; (f) heteroaromatic carbonyl chlorides, triethylamine, CH2Cl2, 50 1C, overnight; (g) 1 N NaOH, MeOH, 90 1C, overnight for 7a–b, 1 N NaOH, MeOH, 60 1C, overnight for 7c and 1 N LiOH, MeOH, 60 1C, 1 h for 7d.
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4-tetrahydroisoquinoline-2-yl)ethyl)aniline (3) in 82% yield (Elslager et al., 1972). Compound 3 was coupled with 3-nitro4-carbomethoxybenzyl chloride to afford methyl 4-((4-(2-(6, 7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl) aminocarbonyl)-2-nitrobenzoate (4) in 88% yield. Compound 4 was reduced to methyl 2-amino-4-((4-(2-(6,7-dimethoxy1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl) benzoate (5) by hydrogenation in the presence of 10% Pd/C at room temperature (RT) in 52% yield. Compound 5 was coupled with different heteroaromatic carbonyl chlorides to yield the standards 6a–d in 53%, 32%, 86% and 67% yield, respectively. Compounds 7a–d were obtained via hydrolysis of 6a–d in MeOH with either 1 N NaOH or 1 N LiOH base and different temperature. For 7a–c, it used 1 N NaOH. At 90 1C, the yields were 63%, 50% for 7a and 7b, respectively. At 60 1C, the yield for 7c was 83%. For 7d, it needed to use 1 N LiOH, and the yield was 74% yield at 60 1C. 2.2. Radiochemistry The radiosynthesis route of carbon-11-labeled tariquidar derivatives [11C]6a–d is displayed in Scheme 2. Acid precursors 7a–d were labeled by a reactive [11C]methylating agent [11C]CH3OTf (Jewett, 1992; Mock et al., 1999) prepared from [11C]CO2, under basic conditions (2 N NaOH) in acetonitrile through the O-[11C]methylation. The radiolabeling reaction mixture was isolated by a simplified solid-phase extraction (SPE) method (Zheng and Mulholland, 1996) to provide target tracers [11C]6a–d in 50–60% radiochemical yields, decay corrected to end of bombardment (EOB), based on [11C]CO2. The large polarity difference between the acid precursor and the labeled O-methylated ester product permitted the use of SPE technique for purification of the labeled product from the radiolabeling reaction mixture. Either a C-18 Plus Sep-Pak cartridge (disposable) or a semi-prep C-18 guard cartridge column (repeat use) was used in SPE purification procedure. The crude reaction mixture was treated with sodium bicarbonate and loaded onto the cartridge by gas pressure. Any non-reacted acid precursor was actually converted to the corresponding sodium salt, and any nonreacted [11C]CH3OTf was actually hydrolyzed to [11C]CH3OH, which would not stick to the C-18 Sep-Pak. The cartridge was washed with water to remove non-reacted [11C]CH3OTf, acid precursor and reaction solvent, and then the final labeled product was eluted with ethanol. Overall synthesis time was 15–20 min from EOB. SPE technique is fast, efficient and convenient and works very well for the O-methylated ester tracer purification using the acid precursor for radiolabeling (Zheng and Mulholland, 1996). The radiosynthesis was performed in an automated multipurpose 11C-radiosynthesis module, allowing measurement of specific activity during synthesis (Mock et al., 2005a,b). The specific activity was estimated in a range 111–185 GBq/mmol at the end of synthesis (EOS) based on other compounds produced using the same targetry conditions which have been measured by the on-the-fly technique (Mock et al., 2005a; Zheng et al., 2007).
The actual measurement of specific activity at EOS by analytical high performance liquid chromatography (HPLC) (Zheng and Mock, 2005) is in agreement with this estimation. Chemical purity and radiochemical purity were determined by analytical HPLC (Zheng and Mock, 2005). The chemical purity of the precursors and reference standards was 496%. The radiochemical purity of the target tracers was 499% determined by radio-HPLC through g-ray (PIN diode) flow detector, and the chemical purity of the target tracers was 493% determined by reversed-phase HPLC through UV flow detector.
3. Experimental 3.1. General All commercial reagents and solvents were obtained commercially from Sigma-Aldrich and Fisher and used without further purification. [11C]CH3OTf was prepared according to a literature procedure (Mock et al., 1999). Melting points were determined on a MEL-TEMP II capillary tube apparatus and were uncorrected. 1H NMR spectra were recorded on a Bruker Avance II 500 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shifts are expressed in parts per million (ppm, d scale), and coupling constants (J) are in hertz (Hz). Liquid chromatography/mass spectra (LC/MS) analysis was performed on an Agilent system, consisting of an 1100 series HPLC connected to a diode array detector, and a 1946D mass spectrometer configured for positive-ion/negative-ion electrospray ionization. High resolution mass spectra (HRMS) were obtained using a Thermo MAT 95XP-Trap spectrometer. Thin-layer chromatography (TLC) was performed using Analtech silica gel GF254 plates and visualized by UV light. Normal phase flash chromatography was carried out on EM Science silica gel 60 (230–400 mesh) with a forced flow of the indicated solvent system in the proportions described below. All moisture- and/or air-sensitive reactions were performed under a positive pressure of nitrogen maintained by a direct line from a nitrogen source. Analytical HPLC was performed using a Prodigy (Phenomenex) 5 mm C-18 column, 4.6 250 mm; 3:1:1 CH3CN:MeOH:20 mM, pH 6.7 phosphate (buffer solution) mobile phase; flow rate 1.5 mL/min; and UV (254 nm) and g-ray (PIN diode) flow detectors. C-18 Plus Sep-Pak cartridges (WAT020515) were obtained from Waters Corporate Headquarters, Milford, MA. Semi-prep C-18 guard cartridge column 1 1 cm was obtained from E.S. Industries, Berlin, NJ, and part number 300121-C18-BD 10 mm. Sterile Millex-GS 0.22 mm vented filter unit was obtained from Millipore Corporation, Bedford, MA. 3.2. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline (1) Formic acid (140 mL) was added slowly to 2-(3,4-dimethoxyphenyl)ethylamine (50.9 g, 281 mmol) at 0 1C. After stirring at 0 1C for 5 min, paraformaldehyde (8.4 g, 281 mmol) was added,
Scheme 2. Synthesis of carbon-11-labeled tariquidar derivatives [11C]6a–d. Reagents and conditions: (a) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 1C, 3 min.
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and the resulting solution was heated at 50 1C with stirring overnight. Excess formic acid was evaporated under reduced pressure, and the residue was poured into ice-water and basified with 1 N NaOH to pH410. The mixture was extracted with CH2Cl2. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The solid was recrystallized from CH2Cl2 to give 1 (51.7 g, 95%) as a pale yellow solid, mp 78–79 1C (Okano et al., 2006, mp 77.7–78.8 1C). 1H NMR (CDCl3): d 6.57 (s, 1H), 6.50 (s, 1H), 3.96 (s, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.14 (t, 2H, J= 6.0 Hz), 3.09 (s, 1H, NH), 2.74 (t, 2H, J= 6.0 Hz).
3.3. 6,7-Dimethoxy-2-(4-nitrophenethyl)-1,2,3,4tetrahydroisoquinoline (2) A mixture of 4-nitrophenethyl bromide (9.2 g, 40 mmol), compound 1 (8.5 g, 44 mmol) and K2CO3 (12.2 g, 88 mmol) in DMF (100 mL) was heated at 100 1C for 8 h. After cooling to rt, the resulting mixture was poured into ice-water and extracted with CH2Cl2. The combined organic layer was washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography with CH2Cl2/ MeOH (20:1) to give 2 (8.2 g, 64%) as a yellow solid, mp 108– 109 1C (Hubensack, 2005, mp 115 1C). 1H NMR (CDCl3): d 8.16 (d, 2H, J= 9.0 Hz), 7.42 (d, 2H, J= 8.5 Hz), 6.61 (s, 1H), 6.53 (s, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.73 (s, 2H), 3.08 (t, 2H, J =6.0 Hz), 2.74 (t, 2H, J= 7.5 Hz), 2.89–2.84 (m, 4H).
3.4. 4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)aniline (3) To a cooled solution of SnCl2 (13.1 g, 69 mmol) in concentrated HCl (40 mL) was added compound 2 (8.0 g, 23 mmol) portionwise with stirring. After a thick paste formed, MeOH (50 mL) was added. The reaction mixture was stirred and heated at 90 1C for 5 h. MeOH was evaporated, and the mixture was basified with 6 N NaOH to pH410, and extracted with EtOAc. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 3 (6.0 g, 82%) as a pale yellow solid, mp 120–121 1C (Hubensack, 2005, mp 125 1C). 1H NMR (CDCl3): d 7.03 (d, 2H, J= 8.5 Hz), 6.64–6.63 (d, 2H, J= 8.5 Hz), 6.60(s, 1H), 6.53 (s, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.71 (s, 2H), 2.87–2.84 (m, 6H), 2.79–2.76 (m, 2H).
3.5. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2nitrobenzoate (4) To a solution of compound 3 (5.0 g, 16 mmol) in CH2Cl2 (50 mL) was added triethylamine (6.7 mL, 48 mmol), followed by 3-nitro4-carbomethoxybenzyl chloride (5.84 g, 24 mmol). The mixture was stirred and heated at 50 1C for 4 h. After cooling to rt, the reaction mixture was washed with saturated Na2CO3, dried over anhydrous Na2SO4, filtered and concentrated. The solid was recrystallized from MeOH to give 4 (7.4 g, 88%) as a pale yellow ¨ solid, mp 101–102 1C (Kuhnle et al., 2009, mp 101 1C). 1H NMR (CDCl3): d 8.85 (s, 1H), 8.38 (s, 1H), 8.15 (d, 1H, J= 8.0 Hz), 7.69 (d, 1H, J= 8.0 Hz), 7.56 (d, 2H, J =8.0 Hz), 7.18 (d, 2H, J= 8.5 Hz), 6.57 (s, 1H), 6.50 (s, 1H), 3.91 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.65 (s, 2H), 2.90–2.73 (m, 8H).
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3.6. Methyl 2-amino-4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)benzoate (5) A solution of compound 4 (7.0 g, 13.5 mmol) in THF (50 mL) and MeOH (15 mL) was hydrogenated at a pressure of 72 psi in the presence of 10% Pd/C (36 mg) at RT for 7 h. The catalyst was filtered off through celite. The solvent was evaporated, and the residue was purified by column chromatography with CH2Cl2/ MeOH (100:3) to give 5 (3.4 g, 52%) as a pale yellow solid, mp ¨ 103–104 1C (Kuhnle et al., 2009, mp 104–106 1C). 1H NMR (DMSOd6): d 10.22 (s, 1H), 7.81 (d, 1H, J=8.5 Hz), 7.67 (d, 2H, J =8.5 Hz), 7.30 (d, 1H, J= 1.0 Hz), 7.23 (d, 2H, J=8.0 Hz), 7.03 (dd, 1H, J =1.5, 8.0 Hz), 6.84 (s, 2H), 6.66 (s, 1H), 6.64 (s, 1H), 3.82 (s, 3H), 3.70 (s, 3H), 3.69 (s, 3H), 3.58 (s, 2H), 2.81–2.73 (m, 8H). 3.7. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoline-2-carbonylamino)benzoate (6a) To a solution of compound 5 (350 mg, 0.70 mmol) in CH2Cl2 (5 mL) was added triethylamine (0.29 mL, 2.09 mmol), followed by quinoline-2-carbonyl chloride (199 mg, 1.04 mmol). The mixture was stirred and heated at 50 1C overnight. The reaction mixture was concentrated and the residue was washed with MeOH to give ¨ 6a (243 mg, 53%) as a white solid, mp 216 1C (dec.) (Kuhnle et al., 2009, mp 176 1C (dec.)). 1H NMR (DMSO-d6): d 13.11 (s, 1H), 10.47 (s, 1H), 9.39 (d, 1H, J= 1.5 Hz), 8.69 (d, 1H, J=8.5 Hz), 8.33 (d, 1H, J= 8.5 Hz), 8.21 (dd, 2H, J= 4.5, 8.5 Hz), 8.16 (d, 1H, J=8.0 Hz), 7.98– 7.95 (m, 1H), 7.81–7.76 (m, 2H), 7.71 (d, 2H, J= 8.5 Hz), 7.28–7.23 (m, 2H), 6.67 (s, 1H), 6.65 (s, 1H), 4.05 (s, 3H), 3.70 (s, 3H), 3.70 (s, 3H), 3.61 (s, 2H), 2.84–2.74 (m, 8H). LC/MS (m/z, ESI): calculated for C38H37N4O6 ([M+ H] + ) 645.2, found 645.2. 3.8. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl) ethyl)phenyl)aminocarbonyl)-2(quinoline-3-carbonylamino)benzoate (6b) Same procedure as for 6a was performed with compound 5 (400 mg, 0.80 mmol), triethylamine (0.33 mL, 2.38 mmol) and quinoline-3-carbonyl chloride (228 mg, 1.19 mmol) to give 6b ¨ (166 mg, 32%) as a white solid, mp 210 1C (dec.) (Kuhnle et al., 2009, mp 191 1C (dec.)). 1H NMR (DMSO-d6): d 11.54 (s, 1 H), 10.46 (s, 1 H), 9.41 (d, 1H, J= 2.5 Hz), 8.90 (d, 1H, J= 2.0 Hz), 8.78 (d, 1H, J= 1.5 Hz), 8.22 (d, 1H, J= 7.5 Hz), 8.15 (d, 1H, J= 8.5 Hz), 8.09 (d, 1H, J =8.5 Hz), 7.96–7.93 (m, 1H), 7.85 (dd, 1H, J= 1.5, 8.5 Hz), 7.78–7.75 (m, 1H), 7.70 (d, 2H, J= 8.5 Hz), 7.26 (d, 2H, J =8.5 Hz), 6.66 (s, 1H), 6.64 (s, 1H), 3.90 (s, 3H), 3.70 (s, 3H), 3.70 (s, 3H), 3.57 (s, 2H), 2.82–2.72 (m, 8H). LC/MS (m/z, ESI): calculated for C38H37N4O6 ([M+H] + ) 645.2, found 645.2. 3.9. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoline-6-carbonylamino)benzoate (6c) Same procedure as for 6a was performed with compound 5 (400 mg, 0.80 mmol), triethylamine (0.33 mL, 2.38 mmol) and quinoline-6-carbonyl chloride (228 mg, 1.19 mmol) to give 6c ¨ (438 mg, 86%) as a pale yellow solid, mp 191 1C (dec.) (Kuhnle et al., 2009, mp 200 1C (dec.)). 1H NMR (DMSO-d6): d 11.62 (s, 1H), 10.46 (s, 1H), 9.06 (s, 1H), 8.90 (s, 1H), 8.69 (s, 1H), 8.60 (d, 1H, J= 8.0 Hz), 8.29–8.22 (m, 2H), 8.12 (d, 1H, J =8.0 Hz), 7.82 (d, 1H, J= 7.5 Hz), 7.71–7.67 (m, 3H), 7.27 (d, 2H, J=8.0 Hz), 6.66 (s, 1H), 6.64 (s, 1H), 3.92 (s, 3H), 3.70 (s, 6H), 3.56 (s, 2H), 2.82–2.72 (m,
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8H). LC/MS (m/z, ESI): calculated for C38H37N4O6 ([M+ H] + ) 645.2, found 645.2. 3.10. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoxaline-2-carbonylamino)benzoate (6d) Same procedure as for 6a was performed with 5 (400 mg, 0.80 mmol), triethylamine (0.33 mL, 2.38 mmol) and quinoxaline2-carbonyl chloride (230 mg, 1.19 mmol) to give 6d (346 mg, 67%) ¨ as a pale yellow solid, mp 219 1C (dec.) (Kuhnle et al., 2009, mp 163 1C (dec.)). 1H NMR (DMSO-d6): d 12.92 (s, 1H), 10.48 (s, 1H), 9.65(s, 1H), 9.34 (s, 1H), 8.27–8.25 (m, 2H), 8.21 (d, 1H, J=8.5 Hz), 8.07–8.06 (m, 2H), 7.80 (d, 1H, J=8.0 Hz), 7.71 (d, 2H, J= 8.0 Hz), 7.27 (d, 2H, J= 8.0 Hz), 6.66 (s, 1H), 6.65 (s, 1H), 4.05 (s, 3H), 3.70 (s, 6H), 3.58 (s, 2H), 2.83–2.73 (m, 8H). LC/MS (m/z, ESI): calculated for C37H36N5O6 ([M+ H] + ) 646.3, found 646.3. 3.11. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-2carbonylamino)benzoic acid (7a) To a suspension of compound 6a (100 mg, 0.16 mmol) in MeOH (2 mL) was added 1 N NaOH (1.5 mL), and the mixture was heated at 90 1C overnight. MeOH was evaporated, and the mixture was acidified with 1 N HCl. The solid was collected by filtration and washed with EtOAc/Et2O (1:1) to give 7a (61 mg, 63%) as a pale yellow solid, mp 210–211 1C. 1H NMR (DMSO-d6): d 13.51 (s, 1H), 10.55 (s, 1H), 9.42 (s, 1H), 8.69 (d, 1H, J= 8.5 Hz), 8.32 (d, 1H, J= 8.0 Hz), 8.22 (d, 1H, J =8.0 Hz), 8.16 (d, 2H, J= 7.0 Hz), 7.93–7.93 (m, 1H), 7.80-7.75 (m, 4H), 7.33–7.25 (m, 2 H), 6.84 (s, 1H), 6.80 (s, 1H), 4.32 (s, 2H), 3.74 (s, 6H), 3.42–3.13 (m, 8H). MS (m/z, ESITOF): calculated for C37H35N4O6 ([M+ H] + ) 631.2557, found 631.2564. 3.12. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-3carbonylamino)benzoic acid (7b) Same procedure as for 7a was performed with 6b (80 mg, 0.12 mmol) to give 7b (40 mg, 50%) as a pale yellow solid, mp 240–241 1C. 1H NMR (DMSO-d6): d 12.46 (s, 1H), 10.64 (s, 1H), 10.55 (br s, 1H), 9.42 (d, 1H, J= 2.0 Hz), 9.11 (d, 1H, J= 1.5 Hz), 8.99 (d, 1H, J=2.0 Hz), 8.21–8.18 (m, 2H), 8.16 (d, 1H, J=8.5 Hz), 7.96– 7.93 (m, H), 7.81–7.75 (m, 4H), 7.33 (d, 2H, J= 8.5 Hz), 6.84 (s, 1H), 6.80 (s, 1H), 4.50 (s, 1H), 4.29 (s, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.43–2.73 (m, 8H). MS (m/z, ESI-TOF): calculated for C37H35N4O6 ([M+H] + ) 631.2557, found 631.2526. 3.13. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-6carbonylamino)benzoic acid (7c) To a suspension of compound 6c (100 mg, 0.16 mmol) in MeOH (2 mL) was added 1 N NaOH (0.66 mL), and the mixture was heated at 60 1C overnight. MeOH was evaporated, and the mixture was acidified with 1 N HCl. The solid was collected by filtration and washed with EtOAc/Et2O (1:1) to give 7c (81 mg, 83%) as a pale yellow solid, mp 190 1C (dec.). 1H NMR (DMSO-d6): d 14.54 (s, 1H), 10.40 (s, 1H), 9.21 (s, 1H), 9.03 (s, 1H), 8.67 (s, 1H), 8.54 (d, 1H, J= 7.5 Hz), 8.33 (d, 1H, J= 8.0 Hz), 8.18 (t, 2H, J= 8.0 Hz), 7.78– 7.64 (m, 4H), 7.30 (d, 2H, J= 7.5 Hz), 6.81 (s, 1H), 6.78 (s, 1H), 4.30 (s, 2H), 3.73 (s, 6H), 3.37–3.01 (m, 8H). MS (m/z, ESI-TOF): calculated for C37H35N4O6 ([M+ H] + ) 631.2557, found 631.2558.
3.14. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoxaline-2carbonylamino)benzoic acid (7d) To a suspension of compound 6d (40 mg, 0.06 mmol) in MeOH (1 mL) was added 1 N LiOH (0.25 mL), and the mixture was heated at 60 1C for 1 h. MeOH was evaporated, and the mixture was acidified with 1 N HCl. The solid was collected by filtration and washed with EtOAc/Et2O (1:1) to give 7d (29 mg, 74%) as a pale yellow solid, mp 200 1C (dec.). 1H NMR (DMSO-d6): d 14.11 (s, 1H), 10.48 (s, 1H), 9.62 (s, 1H), 9.36 (s, 1H), 8.24–8.16 (m, 3H), 8.05– 7.99 (m, 2H), 7.77–7.72 (m, 3H), 7.31 (d, 2H, J =8.0 Hz), 6.80 (s, 1H), 6.77 (s, 1H), 4.25 (s, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.38–3.00 (m, 8H). MS (m/z, ESI-TOF): calculated for C36H34N5O6 ([M+H] + ) 632.2509, found 632.2500.
3.15. General method for the preparation of carbon-11-labeled tariquidar derivatives, [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoline-2-carbonylamino)benzoate ([11C]6a), [11C]methyl 4-((4(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3carbonylamino)benzoate ([11C]6b), [11C]methyl 4-((4-(2-(6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-6carbonylamino)benzoate ([11C]6c) and [11C]methyl 4-((4-(2-(6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)aminocarbonyl)-2-(quinoxaline-2carbonylamino)benzoate ([11C]6d) [11C]CO2 was produced by the 14N(p,a)11C nuclear reaction in small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV proton cyclotron on research purity nitrogen ( + 1% O2) in a Siemens radionuclide delivery system (Eclipse RDS-111). The acid precursor 7a, 7b, 7c or 7d (0.1–0.3 mg) was dissolved in CH3CN (300 mL). To this solution was added 2 N NaOH (2 mL). The mixture was transferred to a small reaction vial. No-carrier-added (high specific activity) [11C]CH3OTf that was produced by the gas-phase production method (Mock et al., 1999) from [11C]CO2 through [11C]CH4 and [11C]CH3Br with silver triflate (AgOTf) column was passed into the reaction vial at room temperature until radioactivity reached a maximum (~2 min), and then the reaction vial was sealed and heated at 80 1C for 3 min. The contents of the reaction vial were diluted with NaHCO3 (0.1 M, 1 mL). The reaction tube was connected to either a C-18 Plus Sep-Pak cartridge or a semi-prep C-18 guard cartridge column. The labeled product mixture solution was passed onto the cartridge for SPE purification by gas pressure. The cartridge was washed with H2O (2 3 mL), and the aqueous washing was discarded. The product was eluted from the column with EtOH (2 3 mL), and then passed onto a rotatory evaporator. The solvent was removed by evaporation under vacuum. The labeled product [11C]6a, [11C]6b, [11C]6c or [11C]6d was formulated with saline, sterile-filtered through a sterile vented Millex-GS 0.22 mm cellulose acetate membrane and collected into a sterile vial. Total radioactivity was assayed and the total volume was noted for tracer dose dispensing. The overall synthesis time including SPE purification and formulation was 15–20 min. The radiochemical yields decay corrected to EOB, from [11C]CO2, were 50–60%. Retention times (tR) in the analytical HPLC system were: 2.13, 2.35, 2.61, 2.47 min for precursors 7a–d, respectively, and 4.28, 4.15, 4.54, 4.37 min for carbon-11-labeled products [11C]6a–d, respectively, which were confirmed by co-injection with cold standard solution of 6a–d, respectively.
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4. Conclusion
References
An efficient and convenient synthesis of new carbon-11labeled tariquidar derivatives has been well-developed. The synthetic methodology employed classical organic chemistry including Pictet–Spengler reaction, nucleophilic substitution, reduction and hydrogenation reduction, coupling reaction and hydrolysis to synthesize unlabeled tariquidar derivative standards and precursors. Carbon-11 labeling at oxygen position of the acid precursor through O-[11C]methylation was incorporated efficiently using [11C]CH3OTf, a signature reaction of carbon-11 radiochemistry from our laboratory. Radiosynthesis performed in an in-house automated multi-purpose 11C-radiosynthesis module produced new probes in high radiochemical yields and purities, and good amount of radioactivity that is suitable for the preclinical application in animal studies using PET. The final radioactive carbon-11 products with the higher specific radioactivities in a range of 111–185 GBq/mmol at EOS can be obtained within 20 min from EOB with fast SPE purification and formulation. These chemistry results combined with the reported in vitro ¨ biological data (Kuhnle et al., 2009) encourage further in vivo biological evaluation of carbon-11-labeled tariquidar derivatives as new potential PET agents for imaging of breast cancer resistance protein.
Barry, M., Kell, M.R., 2009. Enhancing the adjuvant treatment of hormone receptor positive breast cancer. Breast J. 15, 194–198. Czernin, J., 2002. FDG-PET in breast cancer: a different view of its clinical usefulness. Mol. Imaging Biol. 4, 35–45. Elslager, E., Clarke, J., Werbel, L.M., Worth, D.F., 1972. Folate antagonists. 3. 2,4diamino-6-(heterocyclic)quinazolines, a novel class of antimetabolites with potent antimalarial and antibacterial activity. J. Med. Chem. 15, 827–836. Escalona, S., Blasco, J.A., Reza, M.M., Andradas, E., Gomez, N., 2009. A systematic review of FDG-PET in breast cancer. Med. Oncol. [Epub ahead of print]. Fowler, J.S., Volkow, N.D., Wang, G.J., Ding, Y.S., Dewey, S.L., 1999. PET and drug research and development. J. Nucl. Med. 40, 1154–1163. Hubensack, M., 2005. Approaches to overcome the blood barrier in the chemotherapy of primary and secondary brain tumors: modulation of pglycoprotein 170 and targeting of the transferring receptor. Doctoral Thesis. University of Regensburg, Germany. Jewett, D.M., 1992. A simple synthesis of [11C]methyl triflate. Int. J. Rad. Appl. Instrum. A 43, 1383–1385. ¨ ¨ ¨ Kuhnle, M., Egger, M., Muller, C., Mahringer, A., Bernhardt, G., Fricker, G., Konig, B., Buschauer, A., 2009. Potent and selective inhibitors of breast cancer resistance protein (ABCG2) derived from the p-glycoprotein (ABCB1) modulator tariquidar. J. Med. Chem. 52, 1190–1197. Liu, S., Hsieh, W.-Y., Jiang, Y., Kim, Y.-S., Sreerama, S.G., Chen, X., Jia, B., Wang, F., 2007. Evaluation of a 99mTc-labeled cyclic RGD tetramer for noninvasive imaging Integrin avb3-positive breast cancer. Bioconjug. Chem. 18, 438–446. Mock, B.H., Mulholland, G.K., Vavrek, M.T., 1999. Convenient gas phase bromination of [11C]methane and production of [11C]methyl triflate. Nucl. Med. Biol. 26, 467–471. Mock, B.H., Glick-Wilson, B.E., Zheng, Q.-H., DeGrado, T.R., 2005a. Automated measurement of specific activity of radiolabeled ligands during synthesis. J. Label. Compd. Radiopharm. 48, S224. Mock, B.H., Zheng, Q.-H., DeGrado, T.R., 2005b. A multi-purpose 11C-radiosynthesis system. J. Label. Compd. Radiopharm. 48, S225. Okano, K., Tokuyama, H., Fukuyama, T., 2006. Total synthesis of (+ )-yatakemycin. J. Am. Chem. Soc. 128, 7136–7137. Pons, F., Duch, J., Fuster, D., 2009. Breast cancer therapy: the role of PET-CT in decision making. Q. J. Nucl. Med. Mol. Imaging 53, 210–228. Wang, M., Mickens, J., Gao, M., Miller, K.D., Sledge, G.W., Hutchins, G.D., Zheng, Q.H., 2009. Design and synthesis of carbon-11-labeled dual aromatase-steroid sulfatase inhibitors as new potential PET agents for imaging of aromatase and steroid sulfatase expression in breast cancer. Steroids 74, 896–905. Zheng, Q.-H., Mulholland, G.K., 1996. Improved synthesis of b-CIT and [11C]b-CIT labeled at nitrogen or oxygen positions. Nucl. Med. Biol. 23, 981–986. Zheng, Q.-H., Mock, B.H., 2005. Purification of carbon-11 PET radiotracers from unlabeled precursors by preparative HPLC and SPE. Biomed. Chromatogr. 19, 671–676. Zheng, Q.-H., Gao, M., Mock, B.H., Wang, S., Hara, T., Nazih, R., Miller, M.A., Receveur, T.J., Lopshire, J.C., Groh, W.J., Zipes, Z.P., Hutchins, G.D., DeGrado, T.R., 2007. Synthesis and biodistribution of new radiolabeled high-affinity choline transporter inhibitors [11C]hemicholinium-3 and [18F]hemicholinium3. Bioorg. Med. Chem. Lett. 17, 2220–2224.
Acknowledgments This work was partially supported by the Susan G. Komen for the Cure, Breast Cancer Research Foundation and Indiana Genomics Initiative (INGEN) of Indiana University, which is supported in part by Lilly Endowment Inc. The authors would like to thank Dr. Bruce H. Mock and Barbara E. Glick-Wilson for their assistance in production of [11C]CH3OTf. 1H NMR spectra were recorded on a Bruker Avance II 500 MHz NMR spectrometer in the Department of Chemistry and Chemical Biology at Indiana University Purdue University Indianapolis (IUPUI), which is supported by a NSF-MRI grant CHE-0619254. The referees’ criticisms and editor’s comments for the revision of the manuscript are greatly appreciated.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Synthesis of carbon-11-labeled tariquidar derivatives as new PET agents for imaging of breast cancer resistance protein (ABCG2) Min Wang a, David X. Zheng a, Michael B. Luo a, Mingzhang Gao a, Kathy D. Miller b, Gary D. Hutchins a, Qi-Huang Zheng a, a b
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, L3-208, Indianapolis, IN 46202, USA Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e in f o
a b s t r a c t
Article history: Received 20 November 2009 Received in revised form 29 January 2010 Accepted 6 February 2010
Carbon-11-labeled tariquidar derivatives were first designed and synthesized as new PET agents for imaging of breast cancer resistance protein. The target tracers were prepared by O-[11C]methylation of their corresponding acid precursors using [11C]CH3OTf under basic conditions and isolated by a simplified solidphase extraction (SPE) method in 50–60% radiochemical yields based on [11C]CO2 and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 15–20 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 111–185 GBq/mmol. & 2010 Elsevier Ltd. All rights reserved.
Keywords: Positron emission tomography Breast cancer resistance protein Tariquidar derivatives Radiotracers Imaging
1. Introduction Breast cancer is the most common cancer diagnosed in women and is the second leading cause of death in women after lung cancer in the United States (Wang et al., 2009). Breast cancer resistance protein 2, a member of the G subfamily of the ATP-binding cassette (ABC) transporter superfamily (ABCG2) which transports a wide variety of substrates, is an attractive target for the development of therapeutic agents for use in breast cancer treatment, since ABCG2 plays an important role in conjunction with multidrug resistance (MDR) and is associated with a new concept of tumor development ¨ and progression (cancer stem cell hypothesis) (Kuhnle et al., 2009). Recently a new series of tariquidar derivatives have been developed as potent and selective inhibitors of ABCG2, and the representative compounds methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino)benzoate (6a), methyl 4-((4-(2-(6,7-dimethoxy-1,2,3, 4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3-carbonylamino)benzoate (6b), methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-6-carbonylamino)benzoate (6c) and methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoxaline-2-carbonylamino)benzoate (6d) exhibited excellent biological activity with nanomolar IC50
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E-mail address: [email protected] (Q.-H. Zheng). 0969-8043/$ - see front matter & 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2010.02.008
values as 60, 119, 179 and 183 nM for ABCG2, respectively, and about 100–500 fold selectivity for ABCG2 over other efflux transport proteins such as a member of the B subfamily of the ABC transporter superfamily (ABCB1, p-glycoprotein 170 or multidrug resistance related protein 1) and a member of the C subfamily of the ABC transporter superfamily (ABCC2, multidrug resistance related ¨ protein 2) (Kuhnle et al., 2009). Most breast cancer patients will survive after conventional treatment modalities such as surgery, radiation therapy, hormone therapy, chemotherapy and/or a combination thereof if the breast cancer can be detected at the early stage (Barry and Kell, 2009). However, these treatments still fail to cure a significant number of patients. Rapid and accurate early detection is highly desirable so that various therapeutic regiments can be given before the primary tumors become widely spread to other organs (Liu et al., 2007). In the diagnosis and treatment of breast cancer, the biomedical imaging technique positron emission tomography (PET) has become a clinically valuable and accepted new medical imaging tool (Pons et al., 2009). PET combining with radiopharmaceutical 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) is widely used in clinical settings to diagnose breast cancer (Escalona et al., 2009). [18F]FDG is the only PET breast cancer imaging agent used clinically at this point in time. However, [18F]FDG is not in all cases satisfactory. [18F]FDG as a PET tracer has limitations including the inability to visualize very small size breast tumors in early stage and the low cellular uptake rate in breast cancer (Czernin, 2002). In addition, only a limited number of PET studies using other radiotracers have been conducted in clinical research to image breast cancer and monitor its response to treatment, due to the
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limited accessibility of radiotracers. These limitations have motivated the development of new breast cancer PET imaging agents. Radiotracer development is a key area for advancement of research and clinical applications of PET in cancer diagnosis and treatment (Fowler et al., 1999). ABCG2 is an attractive imaging target for PET early detection of breast cancer in patient. Our hypothesis is that tariquidar derivatives labeled with a positron emitting radionuclide carbon-11 may enable non-invasive monitoring of ABCG2 expression in breast cancer and breast cancer response to ABCG2 inhibitor therapy using PET imaging techniques. To radiolabel ABCG2 therapeutic agents as diagnostic agents, we have first designed and synthesized carbon-11-labeled tariquidar derivatives, 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquino[11C]methyl line-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino)benzoate ([11C]6a), [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3, 4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3-carbonylamino)benzoate ([11C]6b), [11C]methyl 4-((4-(2(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl) aminocarbonyl)-2-(quinoline-6-carbonylamino)benzoate ([11C]6c) and [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoxaline-2-carbonylamino)benzoate ([11C]6d), as new PET agents for imaging of ABCG2. 2. Results and discussion 2.1. Chemistry Four title compounds 6a–d were served as the reference standards and selected for radiolabeling. These tariquidar derivatives are potent
1099
and selective ABCG2 inhibitors with the O-methyl group. This provides position for making carbon-11 tracers with [11C]methyl iodide ([11C]CH3I) or [11C]methyl triflate ([11C]CH3OTf) (Jewett, 1992; Mock et al., 1999), which could serve as PET agents. Therefore, these compounds can be proposed as tools for PET experiments. The purpose of this study is to convert therapeutic agents to diagnostic agents. The target compounds 6a–d and their corresponding acid precursors, 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-2-carbonylamino) benzoic acid (7a), 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3-carbonylamino)benzoic acid (7b), 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-6-carbonylamino)benzoic acid (7c) and 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoxaline-2-carbonylamino)benzoic acid (7d), were synthesized according to the procedures outlined in Scheme 1. Synthesis of the reference standards was based on the literature methods described ¨ previously (Hubensack, 2005; Kuhnle et al., 2009) with slight modifications. Isoquinoline 1 was obtained by the Pictet– Spengler reaction of 2-(3,4-dimethoxyphenyl)ethylamine with paraformaldehyde in formic acid in 95% yield (Okano et al., 2006). Subsequent nucleophilic substitution of compound 1 with 4-nitrophenethyl bromide in the presence of K2CO3 in DMF provided 6,7-dimethoxy-2-(4-nitrophenethyl)-1,2,3,4tetrahydroisoquinoline (2) in 64% yield. Reduction of nitro group of compound 2 was performed efficiently with SnCl2 and concentrated HCl instead of hydrogenation under high pressure. Using this method, it afforded 4-(2-(6,7-dimethoxy-1,2,3,
Scheme 1. Synthesis of tariquidar derivative standards (6a–d) and precursors (7a–d). Reagents and conditions: (a) (CH2O)n, HCO2H, 50 1C, overnight; (b) 4-nitrophenethyl bromide, K2CO3, DMF, 100 1C, 8 h; (c) SnCl2, HCl, MeOH, 90 1C, 5 h; (d) 3-nitro-4-carbomethoxybenzyl chloride, triethylamine, CH2Cl2, 50 1C, 4 h; (e) H2, 10% Pd/C, THFMeOH, RT, 7 h; (f) heteroaromatic carbonyl chlorides, triethylamine, CH2Cl2, 50 1C, overnight; (g) 1 N NaOH, MeOH, 90 1C, overnight for 7a–b, 1 N NaOH, MeOH, 60 1C, overnight for 7c and 1 N LiOH, MeOH, 60 1C, 1 h for 7d.
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4-tetrahydroisoquinoline-2-yl)ethyl)aniline (3) in 82% yield (Elslager et al., 1972). Compound 3 was coupled with 3-nitro4-carbomethoxybenzyl chloride to afford methyl 4-((4-(2-(6, 7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl) aminocarbonyl)-2-nitrobenzoate (4) in 88% yield. Compound 4 was reduced to methyl 2-amino-4-((4-(2-(6,7-dimethoxy1,2,3,4-tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl) benzoate (5) by hydrogenation in the presence of 10% Pd/C at room temperature (RT) in 52% yield. Compound 5 was coupled with different heteroaromatic carbonyl chlorides to yield the standards 6a–d in 53%, 32%, 86% and 67% yield, respectively. Compounds 7a–d were obtained via hydrolysis of 6a–d in MeOH with either 1 N NaOH or 1 N LiOH base and different temperature. For 7a–c, it used 1 N NaOH. At 90 1C, the yields were 63%, 50% for 7a and 7b, respectively. At 60 1C, the yield for 7c was 83%. For 7d, it needed to use 1 N LiOH, and the yield was 74% yield at 60 1C. 2.2. Radiochemistry The radiosynthesis route of carbon-11-labeled tariquidar derivatives [11C]6a–d is displayed in Scheme 2. Acid precursors 7a–d were labeled by a reactive [11C]methylating agent [11C]CH3OTf (Jewett, 1992; Mock et al., 1999) prepared from [11C]CO2, under basic conditions (2 N NaOH) in acetonitrile through the O-[11C]methylation. The radiolabeling reaction mixture was isolated by a simplified solid-phase extraction (SPE) method (Zheng and Mulholland, 1996) to provide target tracers [11C]6a–d in 50–60% radiochemical yields, decay corrected to end of bombardment (EOB), based on [11C]CO2. The large polarity difference between the acid precursor and the labeled O-methylated ester product permitted the use of SPE technique for purification of the labeled product from the radiolabeling reaction mixture. Either a C-18 Plus Sep-Pak cartridge (disposable) or a semi-prep C-18 guard cartridge column (repeat use) was used in SPE purification procedure. The crude reaction mixture was treated with sodium bicarbonate and loaded onto the cartridge by gas pressure. Any non-reacted acid precursor was actually converted to the corresponding sodium salt, and any nonreacted [11C]CH3OTf was actually hydrolyzed to [11C]CH3OH, which would not stick to the C-18 Sep-Pak. The cartridge was washed with water to remove non-reacted [11C]CH3OTf, acid precursor and reaction solvent, and then the final labeled product was eluted with ethanol. Overall synthesis time was 15–20 min from EOB. SPE technique is fast, efficient and convenient and works very well for the O-methylated ester tracer purification using the acid precursor for radiolabeling (Zheng and Mulholland, 1996). The radiosynthesis was performed in an automated multipurpose 11C-radiosynthesis module, allowing measurement of specific activity during synthesis (Mock et al., 2005a,b). The specific activity was estimated in a range 111–185 GBq/mmol at the end of synthesis (EOS) based on other compounds produced using the same targetry conditions which have been measured by the on-the-fly technique (Mock et al., 2005a; Zheng et al., 2007).
The actual measurement of specific activity at EOS by analytical high performance liquid chromatography (HPLC) (Zheng and Mock, 2005) is in agreement with this estimation. Chemical purity and radiochemical purity were determined by analytical HPLC (Zheng and Mock, 2005). The chemical purity of the precursors and reference standards was 496%. The radiochemical purity of the target tracers was 499% determined by radio-HPLC through g-ray (PIN diode) flow detector, and the chemical purity of the target tracers was 493% determined by reversed-phase HPLC through UV flow detector.
3. Experimental 3.1. General All commercial reagents and solvents were obtained commercially from Sigma-Aldrich and Fisher and used without further purification. [11C]CH3OTf was prepared according to a literature procedure (Mock et al., 1999). Melting points were determined on a MEL-TEMP II capillary tube apparatus and were uncorrected. 1H NMR spectra were recorded on a Bruker Avance II 500 MHz NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shifts are expressed in parts per million (ppm, d scale), and coupling constants (J) are in hertz (Hz). Liquid chromatography/mass spectra (LC/MS) analysis was performed on an Agilent system, consisting of an 1100 series HPLC connected to a diode array detector, and a 1946D mass spectrometer configured for positive-ion/negative-ion electrospray ionization. High resolution mass spectra (HRMS) were obtained using a Thermo MAT 95XP-Trap spectrometer. Thin-layer chromatography (TLC) was performed using Analtech silica gel GF254 plates and visualized by UV light. Normal phase flash chromatography was carried out on EM Science silica gel 60 (230–400 mesh) with a forced flow of the indicated solvent system in the proportions described below. All moisture- and/or air-sensitive reactions were performed under a positive pressure of nitrogen maintained by a direct line from a nitrogen source. Analytical HPLC was performed using a Prodigy (Phenomenex) 5 mm C-18 column, 4.6 250 mm; 3:1:1 CH3CN:MeOH:20 mM, pH 6.7 phosphate (buffer solution) mobile phase; flow rate 1.5 mL/min; and UV (254 nm) and g-ray (PIN diode) flow detectors. C-18 Plus Sep-Pak cartridges (WAT020515) were obtained from Waters Corporate Headquarters, Milford, MA. Semi-prep C-18 guard cartridge column 1 1 cm was obtained from E.S. Industries, Berlin, NJ, and part number 300121-C18-BD 10 mm. Sterile Millex-GS 0.22 mm vented filter unit was obtained from Millipore Corporation, Bedford, MA. 3.2. 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline (1) Formic acid (140 mL) was added slowly to 2-(3,4-dimethoxyphenyl)ethylamine (50.9 g, 281 mmol) at 0 1C. After stirring at 0 1C for 5 min, paraformaldehyde (8.4 g, 281 mmol) was added,
Scheme 2. Synthesis of carbon-11-labeled tariquidar derivatives [11C]6a–d. Reagents and conditions: (a) [11C]CH3OTf, CH3CN, 2 N NaOH, 80 1C, 3 min.
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and the resulting solution was heated at 50 1C with stirring overnight. Excess formic acid was evaporated under reduced pressure, and the residue was poured into ice-water and basified with 1 N NaOH to pH410. The mixture was extracted with CH2Cl2. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The solid was recrystallized from CH2Cl2 to give 1 (51.7 g, 95%) as a pale yellow solid, mp 78–79 1C (Okano et al., 2006, mp 77.7–78.8 1C). 1H NMR (CDCl3): d 6.57 (s, 1H), 6.50 (s, 1H), 3.96 (s, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.14 (t, 2H, J= 6.0 Hz), 3.09 (s, 1H, NH), 2.74 (t, 2H, J= 6.0 Hz).
3.3. 6,7-Dimethoxy-2-(4-nitrophenethyl)-1,2,3,4tetrahydroisoquinoline (2) A mixture of 4-nitrophenethyl bromide (9.2 g, 40 mmol), compound 1 (8.5 g, 44 mmol) and K2CO3 (12.2 g, 88 mmol) in DMF (100 mL) was heated at 100 1C for 8 h. After cooling to rt, the resulting mixture was poured into ice-water and extracted with CH2Cl2. The combined organic layer was washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography with CH2Cl2/ MeOH (20:1) to give 2 (8.2 g, 64%) as a yellow solid, mp 108– 109 1C (Hubensack, 2005, mp 115 1C). 1H NMR (CDCl3): d 8.16 (d, 2H, J= 9.0 Hz), 7.42 (d, 2H, J= 8.5 Hz), 6.61 (s, 1H), 6.53 (s, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.73 (s, 2H), 3.08 (t, 2H, J =6.0 Hz), 2.74 (t, 2H, J= 7.5 Hz), 2.89–2.84 (m, 4H).
3.4. 4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)aniline (3) To a cooled solution of SnCl2 (13.1 g, 69 mmol) in concentrated HCl (40 mL) was added compound 2 (8.0 g, 23 mmol) portionwise with stirring. After a thick paste formed, MeOH (50 mL) was added. The reaction mixture was stirred and heated at 90 1C for 5 h. MeOH was evaporated, and the mixture was basified with 6 N NaOH to pH410, and extracted with EtOAc. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to give 3 (6.0 g, 82%) as a pale yellow solid, mp 120–121 1C (Hubensack, 2005, mp 125 1C). 1H NMR (CDCl3): d 7.03 (d, 2H, J= 8.5 Hz), 6.64–6.63 (d, 2H, J= 8.5 Hz), 6.60(s, 1H), 6.53 (s, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.71 (s, 2H), 2.87–2.84 (m, 6H), 2.79–2.76 (m, 2H).
3.5. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2nitrobenzoate (4) To a solution of compound 3 (5.0 g, 16 mmol) in CH2Cl2 (50 mL) was added triethylamine (6.7 mL, 48 mmol), followed by 3-nitro4-carbomethoxybenzyl chloride (5.84 g, 24 mmol). The mixture was stirred and heated at 50 1C for 4 h. After cooling to rt, the reaction mixture was washed with saturated Na2CO3, dried over anhydrous Na2SO4, filtered and concentrated. The solid was recrystallized from MeOH to give 4 (7.4 g, 88%) as a pale yellow ¨ solid, mp 101–102 1C (Kuhnle et al., 2009, mp 101 1C). 1H NMR (CDCl3): d 8.85 (s, 1H), 8.38 (s, 1H), 8.15 (d, 1H, J= 8.0 Hz), 7.69 (d, 1H, J= 8.0 Hz), 7.56 (d, 2H, J =8.0 Hz), 7.18 (d, 2H, J= 8.5 Hz), 6.57 (s, 1H), 6.50 (s, 1H), 3.91 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.65 (s, 2H), 2.90–2.73 (m, 8H).
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3.6. Methyl 2-amino-4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)benzoate (5) A solution of compound 4 (7.0 g, 13.5 mmol) in THF (50 mL) and MeOH (15 mL) was hydrogenated at a pressure of 72 psi in the presence of 10% Pd/C (36 mg) at RT for 7 h. The catalyst was filtered off through celite. The solvent was evaporated, and the residue was purified by column chromatography with CH2Cl2/ MeOH (100:3) to give 5 (3.4 g, 52%) as a pale yellow solid, mp ¨ 103–104 1C (Kuhnle et al., 2009, mp 104–106 1C). 1H NMR (DMSOd6): d 10.22 (s, 1H), 7.81 (d, 1H, J=8.5 Hz), 7.67 (d, 2H, J =8.5 Hz), 7.30 (d, 1H, J= 1.0 Hz), 7.23 (d, 2H, J=8.0 Hz), 7.03 (dd, 1H, J =1.5, 8.0 Hz), 6.84 (s, 2H), 6.66 (s, 1H), 6.64 (s, 1H), 3.82 (s, 3H), 3.70 (s, 3H), 3.69 (s, 3H), 3.58 (s, 2H), 2.81–2.73 (m, 8H). 3.7. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoline-2-carbonylamino)benzoate (6a) To a solution of compound 5 (350 mg, 0.70 mmol) in CH2Cl2 (5 mL) was added triethylamine (0.29 mL, 2.09 mmol), followed by quinoline-2-carbonyl chloride (199 mg, 1.04 mmol). The mixture was stirred and heated at 50 1C overnight. The reaction mixture was concentrated and the residue was washed with MeOH to give ¨ 6a (243 mg, 53%) as a white solid, mp 216 1C (dec.) (Kuhnle et al., 2009, mp 176 1C (dec.)). 1H NMR (DMSO-d6): d 13.11 (s, 1H), 10.47 (s, 1H), 9.39 (d, 1H, J= 1.5 Hz), 8.69 (d, 1H, J=8.5 Hz), 8.33 (d, 1H, J= 8.5 Hz), 8.21 (dd, 2H, J= 4.5, 8.5 Hz), 8.16 (d, 1H, J=8.0 Hz), 7.98– 7.95 (m, 1H), 7.81–7.76 (m, 2H), 7.71 (d, 2H, J= 8.5 Hz), 7.28–7.23 (m, 2H), 6.67 (s, 1H), 6.65 (s, 1H), 4.05 (s, 3H), 3.70 (s, 3H), 3.70 (s, 3H), 3.61 (s, 2H), 2.84–2.74 (m, 8H). LC/MS (m/z, ESI): calculated for C38H37N4O6 ([M+ H] + ) 645.2, found 645.2. 3.8. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl) ethyl)phenyl)aminocarbonyl)-2(quinoline-3-carbonylamino)benzoate (6b) Same procedure as for 6a was performed with compound 5 (400 mg, 0.80 mmol), triethylamine (0.33 mL, 2.38 mmol) and quinoline-3-carbonyl chloride (228 mg, 1.19 mmol) to give 6b ¨ (166 mg, 32%) as a white solid, mp 210 1C (dec.) (Kuhnle et al., 2009, mp 191 1C (dec.)). 1H NMR (DMSO-d6): d 11.54 (s, 1 H), 10.46 (s, 1 H), 9.41 (d, 1H, J= 2.5 Hz), 8.90 (d, 1H, J= 2.0 Hz), 8.78 (d, 1H, J= 1.5 Hz), 8.22 (d, 1H, J= 7.5 Hz), 8.15 (d, 1H, J= 8.5 Hz), 8.09 (d, 1H, J =8.5 Hz), 7.96–7.93 (m, 1H), 7.85 (dd, 1H, J= 1.5, 8.5 Hz), 7.78–7.75 (m, 1H), 7.70 (d, 2H, J= 8.5 Hz), 7.26 (d, 2H, J =8.5 Hz), 6.66 (s, 1H), 6.64 (s, 1H), 3.90 (s, 3H), 3.70 (s, 3H), 3.70 (s, 3H), 3.57 (s, 2H), 2.82–2.72 (m, 8H). LC/MS (m/z, ESI): calculated for C38H37N4O6 ([M+H] + ) 645.2, found 645.2. 3.9. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoline-6-carbonylamino)benzoate (6c) Same procedure as for 6a was performed with compound 5 (400 mg, 0.80 mmol), triethylamine (0.33 mL, 2.38 mmol) and quinoline-6-carbonyl chloride (228 mg, 1.19 mmol) to give 6c ¨ (438 mg, 86%) as a pale yellow solid, mp 191 1C (dec.) (Kuhnle et al., 2009, mp 200 1C (dec.)). 1H NMR (DMSO-d6): d 11.62 (s, 1H), 10.46 (s, 1H), 9.06 (s, 1H), 8.90 (s, 1H), 8.69 (s, 1H), 8.60 (d, 1H, J= 8.0 Hz), 8.29–8.22 (m, 2H), 8.12 (d, 1H, J =8.0 Hz), 7.82 (d, 1H, J= 7.5 Hz), 7.71–7.67 (m, 3H), 7.27 (d, 2H, J=8.0 Hz), 6.66 (s, 1H), 6.64 (s, 1H), 3.92 (s, 3H), 3.70 (s, 6H), 3.56 (s, 2H), 2.82–2.72 (m,
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8H). LC/MS (m/z, ESI): calculated for C38H37N4O6 ([M+ H] + ) 645.2, found 645.2. 3.10. Methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoxaline-2-carbonylamino)benzoate (6d) Same procedure as for 6a was performed with 5 (400 mg, 0.80 mmol), triethylamine (0.33 mL, 2.38 mmol) and quinoxaline2-carbonyl chloride (230 mg, 1.19 mmol) to give 6d (346 mg, 67%) ¨ as a pale yellow solid, mp 219 1C (dec.) (Kuhnle et al., 2009, mp 163 1C (dec.)). 1H NMR (DMSO-d6): d 12.92 (s, 1H), 10.48 (s, 1H), 9.65(s, 1H), 9.34 (s, 1H), 8.27–8.25 (m, 2H), 8.21 (d, 1H, J=8.5 Hz), 8.07–8.06 (m, 2H), 7.80 (d, 1H, J=8.0 Hz), 7.71 (d, 2H, J= 8.0 Hz), 7.27 (d, 2H, J= 8.0 Hz), 6.66 (s, 1H), 6.65 (s, 1H), 4.05 (s, 3H), 3.70 (s, 6H), 3.58 (s, 2H), 2.83–2.73 (m, 8H). LC/MS (m/z, ESI): calculated for C37H36N5O6 ([M+ H] + ) 646.3, found 646.3. 3.11. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-2carbonylamino)benzoic acid (7a) To a suspension of compound 6a (100 mg, 0.16 mmol) in MeOH (2 mL) was added 1 N NaOH (1.5 mL), and the mixture was heated at 90 1C overnight. MeOH was evaporated, and the mixture was acidified with 1 N HCl. The solid was collected by filtration and washed with EtOAc/Et2O (1:1) to give 7a (61 mg, 63%) as a pale yellow solid, mp 210–211 1C. 1H NMR (DMSO-d6): d 13.51 (s, 1H), 10.55 (s, 1H), 9.42 (s, 1H), 8.69 (d, 1H, J= 8.5 Hz), 8.32 (d, 1H, J= 8.0 Hz), 8.22 (d, 1H, J =8.0 Hz), 8.16 (d, 2H, J= 7.0 Hz), 7.93–7.93 (m, 1H), 7.80-7.75 (m, 4H), 7.33–7.25 (m, 2 H), 6.84 (s, 1H), 6.80 (s, 1H), 4.32 (s, 2H), 3.74 (s, 6H), 3.42–3.13 (m, 8H). MS (m/z, ESITOF): calculated for C37H35N4O6 ([M+ H] + ) 631.2557, found 631.2564. 3.12. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-3carbonylamino)benzoic acid (7b) Same procedure as for 7a was performed with 6b (80 mg, 0.12 mmol) to give 7b (40 mg, 50%) as a pale yellow solid, mp 240–241 1C. 1H NMR (DMSO-d6): d 12.46 (s, 1H), 10.64 (s, 1H), 10.55 (br s, 1H), 9.42 (d, 1H, J= 2.0 Hz), 9.11 (d, 1H, J= 1.5 Hz), 8.99 (d, 1H, J=2.0 Hz), 8.21–8.18 (m, 2H), 8.16 (d, 1H, J=8.5 Hz), 7.96– 7.93 (m, H), 7.81–7.75 (m, 4H), 7.33 (d, 2H, J= 8.5 Hz), 6.84 (s, 1H), 6.80 (s, 1H), 4.50 (s, 1H), 4.29 (s, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.43–2.73 (m, 8H). MS (m/z, ESI-TOF): calculated for C37H35N4O6 ([M+H] + ) 631.2557, found 631.2526. 3.13. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-6carbonylamino)benzoic acid (7c) To a suspension of compound 6c (100 mg, 0.16 mmol) in MeOH (2 mL) was added 1 N NaOH (0.66 mL), and the mixture was heated at 60 1C overnight. MeOH was evaporated, and the mixture was acidified with 1 N HCl. The solid was collected by filtration and washed with EtOAc/Et2O (1:1) to give 7c (81 mg, 83%) as a pale yellow solid, mp 190 1C (dec.). 1H NMR (DMSO-d6): d 14.54 (s, 1H), 10.40 (s, 1H), 9.21 (s, 1H), 9.03 (s, 1H), 8.67 (s, 1H), 8.54 (d, 1H, J= 7.5 Hz), 8.33 (d, 1H, J= 8.0 Hz), 8.18 (t, 2H, J= 8.0 Hz), 7.78– 7.64 (m, 4H), 7.30 (d, 2H, J= 7.5 Hz), 6.81 (s, 1H), 6.78 (s, 1H), 4.30 (s, 2H), 3.73 (s, 6H), 3.37–3.01 (m, 8H). MS (m/z, ESI-TOF): calculated for C37H35N4O6 ([M+ H] + ) 631.2557, found 631.2558.
3.14. 4-((4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)amino-carbonyl)-2-(quinoxaline-2carbonylamino)benzoic acid (7d) To a suspension of compound 6d (40 mg, 0.06 mmol) in MeOH (1 mL) was added 1 N LiOH (0.25 mL), and the mixture was heated at 60 1C for 1 h. MeOH was evaporated, and the mixture was acidified with 1 N HCl. The solid was collected by filtration and washed with EtOAc/Et2O (1:1) to give 7d (29 mg, 74%) as a pale yellow solid, mp 200 1C (dec.). 1H NMR (DMSO-d6): d 14.11 (s, 1H), 10.48 (s, 1H), 9.62 (s, 1H), 9.36 (s, 1H), 8.24–8.16 (m, 3H), 8.05– 7.99 (m, 2H), 7.77–7.72 (m, 3H), 7.31 (d, 2H, J =8.0 Hz), 6.80 (s, 1H), 6.77 (s, 1H), 4.25 (s, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.38–3.00 (m, 8H). MS (m/z, ESI-TOF): calculated for C36H34N5O6 ([M+H] + ) 632.2509, found 632.2500.
3.15. General method for the preparation of carbon-11-labeled tariquidar derivatives, [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4tetrahydroisoquinoline-2-yl)ethyl)phenyl)aminocarbonyl)-2(quinoline-2-carbonylamino)benzoate ([11C]6a), [11C]methyl 4-((4(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-3carbonylamino)benzoate ([11C]6b), [11C]methyl 4-((4-(2-(6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)aminocarbonyl)-2-(quinoline-6carbonylamino)benzoate ([11C]6c) and [11C]methyl 4-((4-(2-(6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline-2yl)ethyl)phenyl)aminocarbonyl)-2-(quinoxaline-2carbonylamino)benzoate ([11C]6d) [11C]CO2 was produced by the 14N(p,a)11C nuclear reaction in small volume (9.5 cm3) aluminum gas target (CTI) from 11 MeV proton cyclotron on research purity nitrogen ( + 1% O2) in a Siemens radionuclide delivery system (Eclipse RDS-111). The acid precursor 7a, 7b, 7c or 7d (0.1–0.3 mg) was dissolved in CH3CN (300 mL). To this solution was added 2 N NaOH (2 mL). The mixture was transferred to a small reaction vial. No-carrier-added (high specific activity) [11C]CH3OTf that was produced by the gas-phase production method (Mock et al., 1999) from [11C]CO2 through [11C]CH4 and [11C]CH3Br with silver triflate (AgOTf) column was passed into the reaction vial at room temperature until radioactivity reached a maximum (~2 min), and then the reaction vial was sealed and heated at 80 1C for 3 min. The contents of the reaction vial were diluted with NaHCO3 (0.1 M, 1 mL). The reaction tube was connected to either a C-18 Plus Sep-Pak cartridge or a semi-prep C-18 guard cartridge column. The labeled product mixture solution was passed onto the cartridge for SPE purification by gas pressure. The cartridge was washed with H2O (2 3 mL), and the aqueous washing was discarded. The product was eluted from the column with EtOH (2 3 mL), and then passed onto a rotatory evaporator. The solvent was removed by evaporation under vacuum. The labeled product [11C]6a, [11C]6b, [11C]6c or [11C]6d was formulated with saline, sterile-filtered through a sterile vented Millex-GS 0.22 mm cellulose acetate membrane and collected into a sterile vial. Total radioactivity was assayed and the total volume was noted for tracer dose dispensing. The overall synthesis time including SPE purification and formulation was 15–20 min. The radiochemical yields decay corrected to EOB, from [11C]CO2, were 50–60%. Retention times (tR) in the analytical HPLC system were: 2.13, 2.35, 2.61, 2.47 min for precursors 7a–d, respectively, and 4.28, 4.15, 4.54, 4.37 min for carbon-11-labeled products [11C]6a–d, respectively, which were confirmed by co-injection with cold standard solution of 6a–d, respectively.
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4. Conclusion
References
An efficient and convenient synthesis of new carbon-11labeled tariquidar derivatives has been well-developed. The synthetic methodology employed classical organic chemistry including Pictet–Spengler reaction, nucleophilic substitution, reduction and hydrogenation reduction, coupling reaction and hydrolysis to synthesize unlabeled tariquidar derivative standards and precursors. Carbon-11 labeling at oxygen position of the acid precursor through O-[11C]methylation was incorporated efficiently using [11C]CH3OTf, a signature reaction of carbon-11 radiochemistry from our laboratory. Radiosynthesis performed in an in-house automated multi-purpose 11C-radiosynthesis module produced new probes in high radiochemical yields and purities, and good amount of radioactivity that is suitable for the preclinical application in animal studies using PET. The final radioactive carbon-11 products with the higher specific radioactivities in a range of 111–185 GBq/mmol at EOS can be obtained within 20 min from EOB with fast SPE purification and formulation. These chemistry results combined with the reported in vitro ¨ biological data (Kuhnle et al., 2009) encourage further in vivo biological evaluation of carbon-11-labeled tariquidar derivatives as new potential PET agents for imaging of breast cancer resistance protein.
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Acknowledgments This work was partially supported by the Susan G. Komen for the Cure, Breast Cancer Research Foundation and Indiana Genomics Initiative (INGEN) of Indiana University, which is supported in part by Lilly Endowment Inc. The authors would like to thank Dr. Bruce H. Mock and Barbara E. Glick-Wilson for their assistance in production of [11C]CH3OTf. 1H NMR spectra were recorded on a Bruker Avance II 500 MHz NMR spectrometer in the Department of Chemistry and Chemical Biology at Indiana University Purdue University Indianapolis (IUPUI), which is supported by a NSF-MRI grant CHE-0619254. The referees’ criticisms and editor’s comments for the revision of the manuscript are greatly appreciated.